Sphingosine kinases (SphK1, SphK2) catalyze the formation of an important extracellular mediator, sphingosine 1-phosphate (S1P). A fundamental aspect of S1P biology is the large difference in S1P abundance between blood or lymph (high) and tissue (low), which is termed the S1P vascular gradient. This gradient maintains vascular endothelial barrier function and facilitates lymphocyte mobilization from lymphoid tissues. Indeed, S1P1 receptor agonist drugs (e.g. fingolimod) are therapeutically beneficial because S1P signaling is highly sensitive to changes in S1P gradient. We used our SphK2 inhibitors to demonstrate that interdicting S1P signaling at the level of synthesis steepens the S1P vascular gradient by slowing S1P clearance from the blood. This result suggests that SphK2 inhibitors will be extremely useful in treating conditions where the endothelial barrier is compromised, e.g. acute kidney injury and sepsis. Although our recently discovered SphK2 inhibitors are active in vivo, improvements in potency, oral availability and chemical diversity are needed to advance them to the clinic. We will accomplish these goals by generating additional inhibitors on our current chemical scaffold and by developing a novel second scaffold. The current scaffold has also yielded a few SphK1 inhibitors but these lack potency at mouse SphK1, which precludes their testing for efficacy in some key disease models. In contrast to SphK2, inhibition of Sphk1 decreases the S1P vascular gradient and to probe the resulting physiological consequences, multiple inhibitors are needed. We will use iterative rounds of synthesis and testing to generate a library of SphK1 inhibitors with emphases on increasing their potency at mouse SphK1 and discovering inhibitors that have suitable pharmacokinetic properties in rodents. To understand the molecular mechanism of SphK inhibition as well as to inform the synthetic chemistry strategies, we will solve the structures of both isozymes with bound inhibitors using X-ray crystallography. Finally, we will discover a blocker of the S1P exporter, SPNS2, which provides the S1P to lymph and thereby maintains the S1P vascular gradient that is required for lymphocyte egress from lymphoid organs to lymph. Currently, due to the unavailability of SPNS2 inhibitors, this particular approach to the manipulation of S1P gradient and subsequent immunomodulation remains completely unexplored. The strength of our program is the synergism in the combination of chemistry (Santos) and pharmacology (Lynch) to which we now add structural biology (Faham). Our central theme of is to understand the therapeutic potential of manipulating the S1P gradients either at the level of synthesis (SphK inhibition) or transport (SPNS2 blockade). We have a track record of productivity that enabled a fundamental insight into S1P biology, e.g. our discovery that SphK2 inhibition modulates S1P signaling to protect endothelial function, a new therapeutic strategy. Now, we propose the development and detailed characterization of greatly improved SphK inhibitors and to make the chemical tools necessary to interrogate SPNS2 as a potential drug target.